EP1149167B1 - Viral vaccine - Google Patents

Abstract

The present invention relates to a pharmaceutical composition or a vaccine which comprises a mixture of viral protein molecules which are sequence variants of a single viral protein or of part of same. The invention furthermore relates inter alia to a DNA vaccine which codes for a mixture of structurally different virus proteins, the vaccine containing a mixture of sequence variants of a viral DNA molecule or of part of same which code for sequence variants of a viral protein or part. According to a preferred version of the invention, the viral proteins are sequence variants of the GP120 protein of the human immunodeficiency virus (HIV) which differ from each another in their amino acid sequence in the region of the V2 loop and/or of the V3 loop. The invention furthermore relates to the preparation of the virus vaccines including the intermediate stages or constructs, preparation processes and uses connected with them.

Description

The present invention relates to a pharmaceutical composition or a vaccine which comprises a mixture of viral protein molecules which are sequence variants of a single viral protein or a part thereof, the mixture containing 10 10 ² sequence variants which by expression of a plasmid DNA Mixture is available which has randomly distributed sequence combinations due to the variation of nucleotide positions. The invention further relates, inter alia, to a DNA vaccine which codes for a mixture of structurally different virus proteins, the vaccine containing a mixture of sequence variants of a viral DNA molecule or a part thereof, which code sequence variants of a viral protein or part, where the mixture contains 10 10 ² DNA molecules which differ from one another in their nucleic acid sequence, the mixture having randomly distributed sequence combinations due to the variation in nucleotide positions. According to a preferred embodiment of the invention, the viral proteins are sequence variants of the GP120 protein of human immunodeficiency virus (HIV), each of which has an amino acid sequence in the region of the V2 loop and / or the V3 loop, preferably both the V2 - as well as the V3 loop. The invention further relates to the production of the virus vaccines, including the related intermediate stages or constructs, production processes and uses.

For many viral infections, especially HIV-1 infection, one observes a strong immune defense, which in the Is able to get the virus over a period of several years check. The period during which the virus is checked and no symptoms of illness are observed than the asymptomatic phase of the (HIV) disease. In the course new variants of the virus keep arising. This enables the virus, the human immune system to escape and keep getting new immune system cells to infect (see M. Schreiber et al., J. Virol. 68 no. 6 (1994) 3908-3916; J. Gen. Virol. 77: 2403-2414 (1996); Clin. Exp. Immunol. 107 (1997) 15-20; J. Virol. 71 No. 12 (1997) 9198 to 9205).

In the prior art for the treatment of viral diseases, such as. B. Poliovirus (Horaud F et al., Biologicals, 1993, 21: 311-316), Hantavirus (Ulrich R et al., 1998 Vaccine 16: 272-280; Schmaljohn CS et al., 1992 Vaccine 10: 10-13), Lassavirus- (Morrison HG et al., 1989 Virology 171: 179-188; Clegg JC et al., 1987 Lancet 2: 186-188), Hepatitis A virus (Clemens et al., 1995 J. Infect. Dis. 171: Suppl1: S44-S49; Andre et al., 1990 Prog. Med. Virol. 37: 72-95) and hepatitis B virus infection (McAleer et al., 1984 Nature 307: 178-180) but also HIV infection (Egan et al., 1995 J. Infect. Dis. 171: 1623-1627, Kovac et al., 1993 J. Clin. Invest. 92: 919-928) only a few, Genetically unchanged, specific virus antigens or individual inactivated virus strains have been used to investigate suitable vaccination strategies. In the case of HIV, for example, the external coat proteins of two virus strains for vaccine experiments in humans have so far been produced (MN and SF2) (Zolla-Pazner et al., J. Infect. Dis. 178 (1998) 1502-1506). Both GP120 molecules differ in the amino acid sequence, but especially in the amino acid sequence of the variable regions, such as the V3 loop (V3 loop). The two virus strains have different phenotypic properties due to the different V3 loop sequences. The HIV-1 MN strain is a virus variant that preferentially infects macrophages and cells that have the viral coreceptor CCR5. In contrast, viruses of the SF2 type preferentially reproduce in T cells and use the viral coreceptor CXCR4. Such viruses are therefore also called T cell trophies (eg HIV strain SF2) or macrophagotrophs (eg HIV strain MN) virus variants. Vaccination experiments were carried out in the chimpanzee with these two GP120 variants. In these experiments it has been shown that an immune response can be induced which not only protects against the two HIV strains MN and SF2, but is also capable of infection with other virus strains but also with HIV-1 patient isolates prevent. In contrast to chimpanzees, only one of the two GP120 variants has been used in humans for vaccination experiments. Both MN GP120 and SF2 GP120 do not provide certain protection against infection by a virus variant such as is found in an HIV-1 infected person (patient isolate or wild-type virus). JA Levy provides an overview of the state of research in connection with vaccination with GP120 coat protein in " HIV and the Pathogenesis of AIDS ", editor: Jay A. Levy, Chapter 15, 2nd Edition, ASM Press Washington, DC, 1998 ).

The disadvantage of the previously discussed or vaccination strategies examined exist among other things in that you are unable with the vaccines used is, the emergence of new virus variants in the course of To prevent virus disease.

The object of the present invention is therefore a vaccine to provide, which is in particular able to Emergence of new virus variants in the course of a virus disease to prevent or significantly reduce and Ways to restrict or prevent the virus escape human immune defenses and always new ones Defense cells of the immune system can infect.

To achieve the object, the in the Subsequent claims expressed items proposed.

The present invention thus relates, inter alia, to a protein vaccine which comprises a mixture of viral protein molecules, the molecules being sequence variants of a single viral protein or a part thereof, the mixture containing 10 10 ² sequence variants which are produced by expression of a plasmid -DNA mixture is available, which has randomly distributed sequence combinations due to the variation of nucleotide positions.

Sequence variants of a protein are according to the invention understood such molecules that one versus a native viral protein or a part (fragment) thereof Have amino acid sequences, with the variants differ from one another in that at least one Amino acid at any point in the sequence or parts the same can be exchanged. preferably the Sequence variants of several amino acid exchanges at different Set up the sequence for the production, but also the Binding virus neutralizing antibodies are responsible. The number and location of the amino acids exchanged depend on this on the amino acid variability of the regions of the gp120 that observed in cell culture adapted and wild-type HIV isolates becomes. According to the invention, the sequence variants have a Heterogeneity at at least two amino acid positions of the Sequence or parts thereof. One is particularly preferred Heterogeneity at three to eight and preferably at more than eight amino acid positions, with all occurring amino acids are possible at these positions. From the combination of the different amino acids that are possible for each position the number of possible sequence variants in their Whole represent the vaccine.

The invention relates to a vaccine which comprises a mixture of 10 10 ² sequence variants, ie a mixture of more than 10 ² molecules of different amino acid sequence (homologues). A vaccine which comprises 10 10 ³ and preferably 10 10 ⁴ sequence variants is particularly preferred.

In the context of the present invention, it is assumed that that the vaccine not only the sequence variants but also the protein as may include such from which the sequence variants are derived are.

According to the invention, an active ingredient against viruses is thus provided, which includes virus-specific proteins, all of which are found in distinguish their sequences or parts thereof. To do this the protein-coding gene is newly synthesized, to create new interfaces for DNA-cutting enzymes, to allow the exchange of specific regions. By chemical synthesis of DNA fragments relevant to the subject Encode protein section, then becomes a gene bank for the Protein coding sequence prepared. The expression vectors, which encode the protein are then called Mixture transfected into cells. For those protein-producing Cells can then mix different viral cells Proteins are isolated which are preferred according to the invention Represents active ingredient.

In the case of HIV as a viral disease, it comes through chronic infection and the associated continuous Damage to the immune system in the course of the disease complete loss of the specific virus defense. to specific virus defenses include neutralizing antibodies, which have the property with certain antigens Structures to enter into a specific, very firm bond. This marks foreign antigens and their interaction with e.g. Virus receptors blocked. Such a specific one Defense against viruses are neutralizing antibodies against the V3 loop of the external GP120 coat protein. Such anti-V3 loop antibodies are able to prevent cell infection. in the Animal model has been shown to vary by administration of a particular monoclonal V3 loop antibody Prevent infection with HIV-1. By giving the same monoclonal antibody it was also possible to cure an existing infection.

In contrast to experimental systems, one observes however, in the natural course of HIV infection, development always new virus variants. So find yourself at a time hundreds of different in a single patient V3 loop variants because the variation of HIV-1 is precisely in the V3 loop is particularly high. The V3 loop is an important dominant antigenic domain of the GP120. Therefore, there will be one against every V3 loop very specific humoral immune response is formed. Result of Induction of a highly specific immune response against the V3-Loop is that neutralizing antibody against the V3-Loop HIV-1 variant A are unable to convert variant B. neutralize and vice versa (Schreiber et al., J. Virol. 68 (1994) 3908-16, Abrahamsson et al., 4 (1990) 107-12).

In the asymptomatic phase of HIV disease, the cell-free virus variants in serum neutralized by antibodies. Virus variants can only be used at a later point in time can be observed in the serum, the autologous neutralization revoke. These V3 loop escape variants will not recognized by V3 loop antibodies and therefore no longer neutralized. All other virus variants in the serum of the Patients are found but are autologous V3 loop antibody detected. This indicates that in Course of the disease virus variants occur against which no V3 loop antibodies exist. Check it out Antibody content of a patient's serum samples via a Period of several years, as can be seen in the serum samples, taken at the beginning of the asymptomatic phase, the V3 loop antibodies against the later stage Detect V3 loop escape variant. So it doesn't come like for other infectious diseases to the classic escape of Pathogen by always new antigenic variation, but to Switch off the neutralizing immune response against one of the viruses replicating in the patient. The observed lack of neutralizing V3 loop antibody is therefore the result a continuous loss of type-specific V3 loop antibodies. By losing the neutralizing Antibodies can multiply the corresponding virus, which leads to Increases in viral load in the patient's serum leads. In the course the disease is also observed to increase Viral load in the lymph node (Chun et al., Nature 387 (1997) 183-188).

A virus that is in the patient against the immune system prevails and develops into the dominant virus variant, must inevitably overcome all antiviral barriers that the Provides immune system. In addition to neutralizing antibodies cytotoxic T cells are also able to multiply viruses to suppress. Cytotoxic T cells cause death HIV infected CD4 + T cells. In addition to the loss of neutralizing The loss of such cytotoxic antibodies also becomes more T cells against HIV-infected cells observed (Zerhoui et al., Thymus 24 (1997) 203-219; Gould et al., Semin Cell Dev Biol 9 (1998) 321-328; Wagner et al., J Gen Virol 74 (1993) 1261-1269; O'Toole et al., AIDS Res Hum Retroviruses 8 (1992) 1361-1368; Shearer et al., 137: 2514-2521 (1986). Hence the Task of a heterologous vaccine, the mixture of many various GP120 envelope proteins of HIV, both neutralizing Antibodies as well as cytotoxic T cells against many to induce or activate different virus variants.

As already stated, the HIV disease is characterized by this from the fact that the virus is constantly changing. This is through the Error rate of the viral enzyme reverse transcriptase causes which errors in the propagation of viral genetic information makes. This creates mutants that are due on the one hand their biological properties and antiviral effects of the human immune system can be selected. As with HIV there are also other pathogens that contain their genetic information without Rectify error correction, leading to the training of many Variants can lead. In addition to hepatitis A, B and C viruses also include Hanta and Lassa viruses. So you watch at Hepatitis B vaccinated individuals that there was approximately 2% vaccination failure comes. In this 2%, hepatitis B is "escape" Variants found that are not characterized by the Allow vaccine-induced immune response to be suppressed.

The emergence of vaccine, therapy and immune escape variants is based on the genetic variability of these viruses (Blum, Int J Clin Lab Res 27 (1997) 213-214; Jongerius et al., Transfusion 38 (1998) 56-59).

It is not yet known in the prior art how one counteracted such loss of virus neutralizing antibodies can be. The strategies adopted so far were only occasionally successful, i.e. in terms of Formation and maintenance of individual variant-specific Antibodies against certain viruses, the loss of type-specific Antibodies against the diversity of the virus in the course of Protein variants formed due to disease can be the so far proposed treatment methods in the form of antiviral Medicines or vaccines based on a memo substance however do not counteract.

By the present invention is a heterogeneous vaccine it is now possible for the first time to neutralize the loss To prevent V3 antibodies or at least prevent this loss to counteract clearly.

On the basis of the present invention, a immuno-constitutive treatment of virus-infected patients, provided in particular by people infected with HIV which activates the immune system in a way that the naturally acquired immune defense against the virus population of the different virus variants regenerated and re-stimulated the virus variants present in the patient to prevent their multiplication.

In short, the invention is based on the concept for one Drug against HIV-1 to produce a mixture of GP120 proteins, these GP120 proteins e.g. either in the V2 or V3 loop or simultaneously in the two variable domains, i.e. the V2 loop and the V3 loop. To achieve this, the gene encoding GP120 be newly synthesized to form new (monovalent) Interfaces for DNA-cutting enzymes that match the specific Exchange of e.g. Allow V2 and V3 region. Through chemical Synthesis of DNA fragments for the V2 loop and the Coding V3-Loop will then be a V2 / V3 library for GP120 manufactured. The GP120 expression vectors will eventually transfected as a mixture into cells, and from these GP120-producing Cells can then mix the different ones GP120 proteins are isolated, which are used as active ingredients Prophylaxis and / or therapy of HIV disease or AIDS can be used.

The consideration underlying the present invention is therefore for immunotherapy or a vaccine to produce many different GP120 molecules against HIV-1, which carry V3 loop sequences (and / or V2 loop sequences), as they also identified in virus variants in patients can be.

The production of a mixture of natural virus variants in the Cell culture system is very complex. It is particularly important to note that the composition of a virus mixture changes because one has to use cells in the cell culture, that of HIV-1 can be infected. Some virus variants, the selective Have advantages, e.g. faster growth rate, are therefore in the cell culture system after just a few days dominate and supplant the other slower virus variants. This is especially the case when the different virus variants found in the peripheral mononuclear Blood cells (PBMC) or in the serum of patients should be isolated. Since it is not possible to have a consistent one Cultivating a mixture of HIV variants can also in this way no corresponding mixture of GP120 proteins are produced or isolated from virus cultures.

Therefore, a genetic engineering approach for the production of HIV-1 variants and GP120 variants was chosen within the scope of the present invention. A cloning system consisting of two vectors was constructed to produce virus variants and recombinant GP120. The central component of both vectors is a new, chemically synthesized HIV-1 coat protein gene (HIV-1 env gene), which codes for the GP120 protein. One vector contains the entire genome of HIV-1. After transfection of this vector into cells, the cells produce infectious virus particles. This enables a mixture of virus variants to be produced since cells can be used which are resistant to HIV-1 infection. Therefore, in such a cell culture system, the composition of the virus mixture cannot change due to the biological properties of the virus variants. The second vector enables the expression of GP120 variants in eukaryotic cells. This vector is used directly for the production of the vaccine (the vaccine).

A special gene construct for HIV-1 env was produced for the genetic engineering and expression of the V3 loop GP120 variants in eukaryotic cells, which is referred to below as a gene cassette. To produce the gene cassette, the entire coding sequence of the env gene was chemically synthesized. With the help of this method, it is possible to change the coding sequence of the gene as desired, whereby new DNA recognition sequences for DNA-cutting restriction enzymes can be incorporated into the env gene sequence. Care must be taken to ensure that the amino acid sequence of the original GP120 (preferably of the strains NL4-3 and PI-932) does not change, whereas new restriction sites arise in the DNA sequence of the env gene. At the same time, according to the invention, interfaces for enzymes which occur several times in the env sequence are removed, so that there is only one interface for a particular restriction enzyme, such as BglII, in each case. According to a preferred embodiment of the invention, ten new, unique (monovalent) interfaces are inserted at intervals of approximately 150 base pairs in the env gene. The new env gene thus generated is also referred to as a gene cassette in the context of the present invention. In principle, the new env fragment is similar to a polylinker.

The interfaces for the restriction enzymes BstEII and BamHI limit the gene cassette preferred according to the invention (SEQ ID NO: 9). To be able to exchange the area of the env gene, are both interfaces only once in the pBSCenvATG expression vector (SEQ ID NO: 10) for gp120 and in the retroviral vector pNL4-3 available. The vector with the one assigned by the depositor Reference number pBSCenv-V3 was issued on January 6, 1999 at the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany, under the Accession number DSM 12612 stored according to the Budapest contract.

By exchanging the gene section delimited by BstEII and BamHI, the newly produced sections of the env gene, which are also delimited by BstEII and BamHI, can be cloned into both vectors. The PstI / BclI V2 loop and the BglII / XbaI V3 loop env fragment are synthesized chemically or enzymatically and then cloned into a standard vector such as pUC 18 or 19. In this vector the fragment is checked by sequencing and then cut from the vector by a Pst / BclI or BglII / XbaI digest and transferred into the pBSCenvATG and then into the pNL4-3 vector. Instead of intermediate cloning into a standard vector (pUC 18, pUC 19 etc.), the PstI / BclI V2 loop and BglII / XbaI V3 loop fragments can also be cloned directly into pBSCenvATG.

Since there is an interface for a restriction enzyme in the gene cassette every approx. 100 base pairs, all areas of the env gene, in particular the V3 loop or V2 loop, can be exchanged for any DNA fragments. The section coding for the V3 loop can be replaced by a BglII-XbaI fragment which has a size of 244 base pairs (cf. SEQ ID NO: 9; nucleotides 708 to 955). The aim is to replace the gene segments that code for the variable loops of the GP120 protein with new DNA fragments. The fragments are produced synthetically and then cloned into the env gene cassette. If you vary the sequence at certain positions in the synthesis of the V2-Loop and V3-Loop DNA fragments, i.e., insert all four nucleotides with equal rights or use the nucleotide inosine at this position, you can create env gene variants with a predetermined variation of the thereby produce the encoded amino acid sequence. The V3 loop sequences of patient isolates are the basis for the preparation of the GP120 mixture. If these variants are cloned into the gp120 gene cassette, the corresponding GP120 proteins with the variable antigenic domains can be expressed in eukaryotic cells. Sequence data from HIV-1 patient isolates are available for the variation of the V3 loop region of the env gene. Patient isolates are virus variants that have been grown in the laboratory directly from patient material, serum or infected cells from the blood. In contrast to HIV-1 viruses, which have been grown in the laboratory for a long time, these viruses have special properties. These viruses are more resistant to neutralizing antibodies and chemokines. In contrast to virus-adapted viruses, patient isolates use different coreceptors for the infection of cells. Since patient isolates are very different from laboratory-adapted viruses, the V2-Loop or V3-Loop sequence data collected from the env genes of such viruses should be used for the preparation of the GP120 mixture. Within the scope of the invention, additional base exchanges are possible in areas outside of the sequence sections coding for V2 and V3.

According to a particular embodiment of the invention, a Protein vaccine is provided which is a mixture of Human immunodeficiency virus (HIV) GP120 proteins, each in their amino acid sequence in the area of the V3 loop and / or the V2 loop.

Within the scope of the present invention, genetic engineering Production of the protein mixture or the protein vaccine Sequence variations preferably in areas of the V3 loop introduced that lie outside of consensus sequences (cf. e.g. M. Schreiber et al., J. Virol. 71 No. 12 (1997) 9198-9205). The consensus sequences are sequence sections, the both between different strains of virus as well as in the formation of virus variants progressive virus disease (HIV disease) in the body in the remain essentially. Such a section is in In the case of the B-subtype HIV-1, the sequence Gly-Pro-Gly-Arg-Ala-Phe (GPGRAF). To the left and right of this sequence motif short 4-10 amino acid long areas that vary widely can. In Fig. 1 this is based on the example of the V3 loop sequence variations represented by patient isolates.

In addition to variations in the area of the V3 loop, the Molecules of the protein vaccine according to the invention also in the field of the V2 loop have sequence variations. In this case, too are changes in the amino acid sequence outside of consensus sequence ranges preferred (see Fig. 2). Furthermore are additional amino acid exchanges in areas outside the V2und V3 loops possible.

In connection with the present invention, the usual one- or three-letter codes to denote the amino acids used. When varying the amino acid sequences there are any within the protein vaccine according to the invention Amino acid exchange possible. In any case, however Amino acids of the viral GP120 sequence or the sequences of the V2 loop and V3 loop preferably replaced by amino acids, which also applies to the corresponding virus variants Sequence positions occur (see Fig. 2, Stand of the sequence data 1997 in: Human Retroviruses and AIDS, A compilation and analysis of nucleic acid and amino acid sequences, Los Alamos National Laboratory, Los Alamos, NM 87545, U.S.A., Editors: B. Korber, B. Hahn, B. Foley, J.W. Mellors, T. Leitner, G. Myers, F. McCutchan, C. Kuiken).

Within the scope of the present invention, a Protein vaccine provided on genetic engineering Pathways are made and sequence variants more specific viral Antigens (proteins) includes. The manufacture of the vaccine will described below using the example of the V3 loop of HIV. For those skilled in the art will understand that that described below Principle of synthesis on other viruses or viral proteins or Parts of their sequences can be transferred.

In short, a mixture of is used as the basis for the vaccine GP120 proteins with variable V3 sequences produced by the Variability of the V3 loop of the GP120 protein modeled on HIV are.

For this purpose, the sequence coding for the GP120 protein is cloned piecewise into the plasmid pUC 18/19. This is done by a polylinker exchange, so that with the possible restriction sites which were previously inserted in the gp120 sequence by silent mutations, all interesting sections are flanked with monovalent restriction sites and can thus be cut out and exchanged later.

Then two homologous nucleic acid oligomers with Made with the help of a DNA synthesizer (length approx. 300 Base pairs). After hybridization, this forms double-stranded DNA fragment with the DNA sequence of the V3 loop. For the production of the double-stranded V3-Loop DNA fragment can use various standard methods from molecular biology be used. In a method, the variables are Inosine introduced and at the corresponding complementary The variables are stranded by a nucleotidic mix (AGCT, AGC, AG, ...) introduced. With this method, the DNA fragment manufactured exclusively chemically. Another one Method, a combination of chemical and enzymatic DNA synthesis, the variables are made up of nucleotide mixtures introduced. The oligonucleotides are then hybridized at complementary ends of a length of 20-30 bases that are not are variable. The synthesis of the completely double-stranded Molecule takes place enzymatically with the help of a DNA polymerase. Both isothermal DNA polymerases (Klenow fragment) or thermostable DNA polymerases can be used (Taq polymerase). If Taq polymerase is used, then larger ones can be used Amounts for cloning the V3-Loop DNA fragment also with Using the polymerase chain reaction. With With the help of these methods, a double-stranded DNA fragment is created manufactured, which is variable at certain positions. This degenerate DNA sequence codes for the corresponding one Variety of V3 loop amino acid sequences to be produced GP120 protein mixture, the protein vaccine.

The mixture of the synthesized V2 fragments has a PstI at the 5 'end and a BclI interface at the 3' end. The mixture of the synthesized V3 fragments has a BglII and a XbaI interface at the 5 'end. With the help of the respective two interfaces, the fragment mixture is cloned into a vector, for example pUC18 delta-env or pUC 18 BstEII-BamHI (FIG. 3). After this cloning, a mixture or a pool of plasmid DNAs of the vector pUC 18 is produced, all of which have the complete BstEII-BamHI env fragment. All plasmid DNAs differ only in the sequence of the env V2 loop or V3 loop. By inserting the V2 or the V3 fragments, the deletion (delta env) of the env gene segment in the pUC18 vector is canceled.

This mixture of the plasmid DNAs with variable env fragments thus produced is described in E. coli transformed and fermented. E amplifies and replicates. coli this mixture of plasmid DNA. All possible variables of these V2 / V3 loop plasmids are generated.

This plasmid pool is then isolated. The env fragment is then cut out from the mixture of the plasmid DNAs by BstEII and BamHI digestion and cloned directly into the vector for the expression of the viral gp120 . This vector has the advantage that the GP120 is also expressed in the eukaryotic cell system.

This pool of BSCenvATG vector DNA with variable gp120 construct is now transfected in Cos or Chinese Hamster Ovary cells (CHO cells). These eukaryotes then express this pool of plasmids, so that the corresponding protein is statistically translated for each variable and then - depending on which eukaryotes are used - glycosylated accordingly. This is followed by the harvest of the proteins with subsequent purification to the finished product (protein mixture or GP120 mixture with variable amino acid sequence).

The method for generating the Protein vaccine according to the invention in FIG. 4 clearly shown.

As already explained, variations in the V2 loop and / or in the V3 loop. The variation of the V2 loop can take place according to the same scheme described for V3.

The protein vaccine according to the invention is produced as described above, using different key constructs, i.e. Nucleic acid intermediates and DNA constructs used for Execution of the invention are essential.

As already explained above, the nucleic acid sequence encoding the GP120 protein is cloned piecewise into pUC 18/19, whereby according to the invention a gp120 sequence is assumed, in which the multiple restriction sites are first modified by silent mutations so that the remaining restriction sites each occurs only once in the sequence. This sequence modification is necessary in order to be able to insert the expression cassette, which is required to generate the sequence variants, in a targeted manner at a very specific location, which is limited by two restriction cleavage sites which occur only once in the sequence. Methods for introducing silent mutations in a nucleic acid sequence are well known to those skilled in the art.

For the present invention, inter alia, a nucleic acid sequence was generated which is derived from the env sequence in SEQ ID NO: 1 or a fragment thereof, and is modified such that it contains only monovalent restriction sites. The modification is preferably carried out by introducing silent mutations. According to a preferred embodiment of the invention, the nucleic acid sequence has the sequence shown in SEQ ID NO: 9. The sequence of the gene cassette may be modified such that it contains the entire env gene or parts of the env gene of virus isolates from patients. Such a gene cassette should preferably contain the env sequence of the patient isolate PI-932 (SEQ ID NO: 11). A preferred gene cassette according to the invention based on the sequence PI-932 is shown in SEQ ID NO: 12.

The present invention also relates to a single-stranded Nucleic acid sequence that for the V3 loop and / or the area or fragments coding for the V2 loop or parts thereof, where in the case of the V3 loop a BglII-XbaI (247 bp) or a BglII-NheI (283 bp) fragment against a modified fragment which is present on at least 6, preferably at 9 to 20 positions, nucleic acid exchanges or carries mutations, and in the case of the V2 loop a PstI-BclI (139 bp) or a PstI-EcoRI (339 bp) fragment against one altered fragment which on at least 6, preferably on 9 up to 20 positions, nucleic acid exchanges or mutations carries, can be exchanged. Here are the nucleotides within a nucleic acid sequence either by Inosine or in each case by a mixture of 2-4 nucleotides replaced. This is explained using the example in FIG. 5. Should on 7 Amino acid positions of the V3 loop 21 different amino acids 1152 variants are combined, then 11 Nucleic acid positions two nucleotides each by chemical Synthesis in the sequence of single-stranded nucleic acids (Oligonucleotides) are introduced (Fig. 5).

The single-stranded nucleic acid sequences are - as above already mentioned - hybridized or synthesized into a double strand.

The present invention therefore also relates to double-stranded DNA, the hybrid of the above single-stranded nucleic acid sequences comprises, each with a nucleic acid sequence (5'-3'-oligomer or 3'-5'-oligomer) to one or more selected Positions contains inosine and the other nucleic acid sequence (3'-5'-oligomer or 5'-3'-oligomer) to those in the later Hybridization corresponding complementary positions each two, three or four of the possible nucleotides (adenine, A; Thymine, T; Guanin, G; Contains cytosine, C). This will Sequences are generated in which the four bases are randomly distributed the respective positions of the single-stranded nucleic acid sequence (DNA) are present, so that the desired combinations of A, T, G or C for the corresponding desired ones Amino acid codons (a codon is made up of a sequence of three Nucleotides formed) are generated. Because of the variation Randomly distributed sequence combinations occur from nucleotide positions. Therefore, e.g. for the combination of (A, C) (ACT) (ACGT) total 2x3x4 = 24 possible DNA sequences, that code for different amino acids. The number of variable positions also determines the number and Position of the introduced inosine in the complementary DNA sequence. The calculation of the heterogeneity of such Construct is shown in Fig. 5.

Since inosine-containing single-stranded DNA is hybridized with oligomers to form the double-stranded DNA, each containing A, T, G or C at the corresponding positions at random, a mixture of double-stranded gp120 sequence or a part (fragment) thereof is obtained, where the nucleic acid sequences are each derived from the env sequence (SEQ ID NO: 1 or 12) and the nucleic acid sequences differ from one another in the region coding for the V2 loop and / or in the region coding for the V3 loop. This means that the nucleic acid sequences contained in the mixture differ from one another in such a way that they code for a mixture of proteins which each have different amino acid sequences in the V2 loop and / or in the V3 loop.

In the context of the invention, at least 10 ² , preferably at least 10 ³ and, according to a particularly preferred embodiment of the invention, at least 10 ⁴ sequence variants can be obtained at the nucleic acid level (DNA level) in this way. This DNA mixture which contains 10 10 ² DNA molecules which differ from one another in their nucleic acid sequence, the mixture having randomly distributed sequence combinations due to the variation in nucleotide positions, is referred to below as the DNA pool, based on the variants of the gp120 sequence this is referred to below as a pool with a variable gp120 construct ( gp120 pool).

Sequence variants of a DNA sequence are part of the present invention understood such molecules that a towards a native viral DNA molecule or part (Fragment) of the same derived nucleic acid sequence, the variants differ from one another in that at least one nucleotide at any point in the sequence can be exchanged. The sequence variants preferably have multiple nucleotide exchanges at different locations on the Sequence on, the number and location of the exchanged Nucleic acids essentially the length of the nucleic acid sequence depends.

When expressing the env gene variants starting from the plasmid-DNA mixture prepared, it can be expected that all possible DNA sequences will be expressed due to the random distribution. Therefore, all sequence variants of the gp120 molecule or a part thereof that are possible on the basis of the predetermined DNA mixture are then formed. The heterogeneity of the gp120 mixture is calculated on the one hand based on the heterogeneity of the DNA sequence and the degeneration of the genetic code for the amino acids (see also FIG. 5).

The heterogeneity of the plasmid-DNA mixture is demonstrated by the DNA sequencing of individual clones. For this purpose, the mixture of the plasmid DNA is transformed into E. coli and individual clones are selected at random and their V2 or V3 loop sequence is determined. A total of approximately 100-200 different clones are to be sequenced. The statistical distribution of the DNA sequences can be used to calculate the distribution of the entire mixture. The method largely corresponds to the method of taking samples, which is used as standard for the quality control of a wide variety of products.

The direct molecular analysis of the heterogeneity of the GP120 protein Mixing is a little more difficult because do not separate individual GP120 molecules from the mixture and have it proven. This is in the nature of things because of itself the individual GP120 variants only in a few amino acids distinguish from each other. By gel electrophoretic separation or a mass spectroscopic analysis can be determined be it a mix or a single Form of the GP120 molecule. If the GP120 mix e.g. used in the animal as a vaccine is that induced in the animal Immune response directly from the number and composition of the GP120 mix dependent. The animal's immune response, the neutralizing antibodies can then be used in virus neutralization tests to be examined. To control one accordingly Cross-variant immune response can be different Patient isolates of HIV-1 used in the neutralization test become. Patient isolates are preferred which are in the ability to differentiate different coreceptors for that To be able to use infection of the target cells. The neutralization potential the GP120 mixture then serves as a quality standard.

The present invention therefore also relates to a protein mixture which comprises GP120 proteins which each have different amino acid sequences in the V2 loop and / or in the V3 loop, the mixture at least 10 ² (preferably at least 10 ³ and according to a particularly preferred embodiment of the invention, contains at least 10 ⁴ ) sequence variants and can be obtained by expressing a plasmid-DNA mixture which, due to the variation in nucleotide positions, has randomly distributed sequence combinations.

As previously mentioned, the mixture of double-stranded DNA that can be obtained by hybridization of inosine-containing single-stranded DNA with randomly distributed A-, T-, G- and / or C-containing single-stranded DNA, ie the pool with variable GP120 construct , transformed and fermented in prokaryotic or eukaryotic host cells, preferably E. coli .

The present invention therefore relates to plasmids which contain inserted double-stranded DNA, the hybrids of the single-stranded, inosine-containing nucleic acid sequence (see above) with the single-stranded nucleic acid sequence (see above) containing a mixture of all four nucleotide variants (A, T, G, C). include. The invention further relates to a vector mixture which comprises a mixture of these plasmids, the nucleic acid sequences of the plasmids differing from one another in the region coding for the V3 loop and / or in the region coding for the V2 loop, the vector Mixture contains at least 10 ² (preferably at least 10 ³ and according to a particularly preferred embodiment of the invention at least 10 ⁴ ) of the plasmids mentioned and it has randomly distributed sequence combinations due to the variation in nucleotide positions. Depending on which host cells are to be used for the expression of the vectors (plasmids), various expression systems and base vectors which are well known to the person skilled in the art are possible (cf. Methods in Enzymology, Vol. 185, Gene Expression Technology, 1991, publisher DV Goeddel, Academic Press , Inc.). The plasmid pUC 18/19 is preferred for expression in E. coli as the basic plasmid into which the nucleic acid sequence variants are cloned. In the context of the present invention, the vector mixtures can thus either in bacterial host cells such as E. coli , or in eukaryotic host cells, preferably from the group consisting of Cos, CHO, or baby hamster kidney cells (BHK cells), or in other host cells.

The present invention thus further relates to E. coli host cells or eukaryotic host cells which are transfected with a vector mixture according to the invention.

Because host cells are transfected with the DNA pool (the vector mixture) are expected to be as large, or at least an almost equally large number of differently transformed ones Host cells emerge like sequence variants in the DNA pool available. There is something special in the manufacture of the vaccine Attention to the yields in the individual cloning steps to lay. The yields of the production of double-stranded DNA fragments, the ligation of the V3 loop and V2 loop DNA fragments in the env gene and the transformation respectively Production of the bacteria and cell clones must be designed that the heterogeneity of the mixture to be achieved is not restricted becomes. This should be illustrated using an example. If e.g. out of a lot of balls that are due to two Distinguish colors, a number of balls should be taken, but both colors with 99.9% probability in this one If there were to be a selection, approximately 13 balls would have to be removed (G. Schreiber, a combinatorial problem from the Genetics; Bioengineering 1988, 2: 32-35). Transferred to the Cloning steps of the HIV vaccine means: If one Heterogeneity of approximately 6000 virus variants is to be generated, would have to do thirteen times in each cloning step many clones are generated. A gene bank of approx. 80,000 clones can be made for the V3 loop. Starting from This gene bank then becomes the expression vectors for the Transfection of the CHO cells is made. During transfection the CHO cells then have to have approximately 80,000 transfection events be achieved. Such a mix of transfected CHO cells then generate the desired amount of 6000 different gp120 variants.

The present invention also relates to a method of manufacture a nucleic acid sequence coding for a viral protein (Expression cassette), in which the sequence is such modified or preferably so many silent mutations introduces that they then at least two and preferably contains only monovalent restriction sites. Especially the sequence preferably contains only monovalent ones Restriction sites. It is preferably that of the nucleic acid sequence encoded protein around GP120, whereby one by silent mutations the env wild-type sequence coding for the GP120 varied.

The present invention furthermore relates to a process for producing the vector mixture according to the invention, which preferably contains a mixture of plasmids whose nucleic acid sequences are in each case in the region coding for the V2 loop and / or in the region coding for the V3 loop Differentiate the random distribution of the bases from one another at the varied nucleotide positions, the plasmids according to the invention being ligated into a (base) vector which can be expressed in host cells (preferably E. coli , Cos, CHO or BHK cells). The (basic) vector is preferably the pUC 18, the pUC 19 or the BSCenvATG vector.

Within the scope of the present invention, a method for the production / production of host cells, preferably selected from the group consisting of E, is also disclosed . coli , Cos, BHK or CHO cells, in which the host cells are transformed with a vector mixture according to the invention which contains a mixture of plasmids whose nucleic acid sequences are in the region coding for the V2 loop and / or in the Distinguish the region coding for the V3 loop from one another by random distribution of the bases at the varied nucleotide positions.

Finally, according to the invention, a method for producing a DNA vaccine is made available for the first time, in which the method according to the invention for producing the vector mixture is carried out, the plasmids according to the invention being applied to host cells (for example human and animal monocytes) by mixing the gp120 - Express proteins. For application, the DNA vaccine can optionally be formulated with pharmaceutically acceptable auxiliaries and / or carriers and, after administration in the organism, enable the sequence variants of viral proteins to be produced.

The present invention thus also relates to a pharmaceutical composition or a DNA vaccine which codes for a mixture of structurally different virus proteins, the vaccine containing a mixture of sequence variants of a viral DNA molecule or a part thereof, ie of DNA molecules whose nucleic acid sequences differ from one another in the region coding for the protein, a part or a fragment thereof. The mixture contains 10 10 ² DNA molecules which differ from one another in their nucleic acid sequence, the mixture having randomly distributed sequence combinations due to the variation in nucleotide positions. According to the invention, the term “structurally different virus proteins” is understood to mean those proteins whose amino acid sequences are derived from the wild-type sequence of the corresponding virus protein, the amino acid sequences differing in that they differ from one another and in comparison to the wild-type sequence distinguish one or more exchanged amino acids at the same or different positions in the sequence. As already stated, the nucleic acid sequences in the vaccine according to the invention preferably code for a mixture of structurally different GP120 proteins from HIV (sequence variants of GP120), a vaccine which contains a mixture of DNA molecules whose nucleic acid sequences differ in that being particularly preferred differentiate the V2 loop coding region and / or in the region coding for the V3 loop from gp120 of HIV-1. In this connection, reference is also made to FIG. 3, in which known structural differences or variations are shown in the V3 loop of GP120.

The DNA vaccine according to the invention is of particular importance in Framework of gene therapy.

Finally, according to the invention, a method for producing a pharmaceutical composition or a protein vaccine is provided for the first time, in which the host cells according to the invention, ie host cells which have been transformed with a vector mixture according to the invention, the vectors each containing plasmids and their nucleic acid sequences differ from each other in the region coding for the V2 loop and / or in the region coding for the V3 loop by random distribution of the bases at the varied nucleotide positions, cultivated under conditions which allow expression of the mixture of viral protein sequence variants. The host cells are preferably bacterial host cells, such as E. coli , or eukaryotic host cells, preferably from the group consisting of Cos, Chinese Hamster Ovary (CHO), or Baby Hamster Kidney cells (BHK cells). ,

1. das Immunsystem aktiviert,

2. die Bildung HIV-1-neutralisierender Antikörper induziert,

3. den Verlust von neutralisierenden Antikörpern verhindert und

4. die Kontrolle über die Stimulation der GP120-spezifischen Immunantwort durch den Wirkstoff übernommen wird.

The present invention thus makes it possible to provide a vaccine made from variable GP120 proteins which are used in the prophylactic and / or therapeutic treatment of HIV infection or AIDS

1. activates the immune system,

2. induces the formation of HIV-1 neutralizing antibodies,

3. prevents the loss of neutralizing antibodies and

4. Control over the stimulation of the GP120-specific immune response is taken over by the active ingredient.

This is preferably done in that even before the Outbreak of AIDS related loss of HIV neutralizing Antibodies have sufficient antigens available stand because of their variety of different amino acid sequences (i.e. their sequence variations) are able to induce the formation of those HIV antibodies that normally, i.e. without appropriate prophylaxis according to the present invention, lost in disease progression go or decrease in concentration. In addition to one preventive treatment also relates to the present invention the therapeutic treatment, the GP120 mixture (Protein vaccine) is used to decrease or decrease Counteract loss of HIV-neutralizing antibodies, by activating the immune system and the formation of new or additional HIV-1 neutralizing antibody is induced.

According to the invention, therefore, the mixture of structurally different viral proteins, the aforementioned sequence variants of a viral protein (preferably GP120) or a part thereof, are used to produce a vaccine for the prevention and / or therapy of a virus infection in humans. Furthermore, the present invention relates to the use of a mixture of DNA molecules which code for 10 10 ² sequence variants of a viral protein or a part thereof, the mixture having randomly distributed sequence combinations due to the variation in nucleotide positions, for producing a vaccine for prevention and / or Therapy of a virus infection in humans. According to a preferred embodiment, the invention relates to the use for producing a vaccine for the prevention and / or therapy of an HIV infection in humans.

The virus infection can be within the scope of the present Invention to deal with any infection in the course replicating virus variants arise from the disease, being as selected protein sequences or protein coding Nucleic acid sequences are those amino acid sequence sections or nucleic acid segments coding for them come into question according to the invention, in the course of which viral Diseases always or frequently variants, i.e. sequence variations, to be watched. It is preferably present sequence variants of the GP120 molecule (at protein level) or the region coding for GP120 (at DNA level) or parts the same, in particular to sequence variants in the V3 loop or in V2 loop, preferably both in the V3 and the V2 loop.

Pharmaceutical compositions are consequently according to the invention or vaccines provided at the immuno-constitutive treatment of virus-infected people Activate the immune system in a way that is natural acquired immune defense against the virus regenerates and is re-stimulated in order to replicate in the patient To prevent virus variants from multiplying. According to one preferred embodiment of the invention will be the first time Vaccines for the immuno-constitutive treatment of HIV-1 infected people provided.

In the context of the present invention, the vaccines either as such, i.e. as DNA and / or protein mixtures without further additives, or together with other active ingredients, such as. Immune stimulants such as interleukin-2, CC and CXC chemokines, and / or pharmaceutically acceptable auxiliary and Carriers are formulated or administered. Usually are the vaccines according to the invention for intravenous administration formulated such as intravenous, intramuscular, subcutaneously. In addition, the vaccines can be oral, mucosal (intravaginal, intrarectal) and transdermal use be formulated.

The present invention has the following advantages:

Through targeted genetic engineering manipulation of the nucleic acid sequence, with the help of a gene cassette, any Variants of a pathogen or a gene of the pathogen produce. The variations preferably concern the areas against the neutralizing antibody or cytotoxic T cells be formed. This allows a vaccine to be in shape a mixture of variants of the pathogen or of Variants of the corresponding antigens of a pathogen produce. The advantages of this concept in relation to HIV as Pathogens lie in the preparation of a mixture of the HIV-GP120, the outer membrane protein of HIV, against which the virus-neutralizing immune response of humans directed. Because in the case of HIV, every patient is exposed to a variety of HIV variants, all of which in the sequence of the GP120 protein is the Use of a variety of GP120 variants as a vaccine from particular advantage, this GP120 mixture for both Immunization of normal healthy people but also for them Therapy of people already infected with HIV can be. When immunized with the GP120 mixture the broadest possible immune response against as many as possible Virus variants induced in humans, which in the ideal case protects against all types of HIV. In therapy with the GP120 mixture can result in the loss of neutralizing antibodies and the loss of the HIV-specific cellular immune response be fought.

According to the invention, it is also possible for the first time to produce gp120 clones which, in the type or variety of the sequence variations observed, correspond to plasma isolates from HIV-infected or AIDS-sufferers (see M. Schreiber et al., J. Virol. 68 No. 6 (1994) 3908-3916). In this context, in contrast to various approaches pursued to date, no isolated GP120 molecule or an antigenic fragment thereof is used for the immunization, but rather a protein mixture which consists of a large number of sequence variants which are characterized by a complete GP120 sequence which also have the correct tertiary structure corresponding to the natural folding of the GP120 molecule. The conformation of the virus variants made available in the vaccine is of importance insofar as it provides sequence and structural variants which are as close as possible in agreement with the GP120 variants detectable in the course of the virus disease with regard to the observed sequence variations and also for binding to CD4 and the coreceptor required conformation is achieved, whereby an effective immune stimulation can be achieved.

The invention provides a mixture of GP120 proteins, which is the protein vaccine. At the same time, the invention provides a mixture of the genes coding for this protein mixture. These genes can be converted into a vector which is suitable for direct use in humans (DNA vaccine) (Ulmer et al., 1995 Ann NY Acad Sci 772: 117-125; Donnelly et al., 1995 Ann NY Acad Sci 1995 772: 40-46). A DNA vaccine has the advantage that the heterogeneity of the mixture of the DNA vectors can be higher than the mixture of the recombinant GP120 proteins. It is easier to produce a high heterogeneity of a DNA-vector mixture. Such a DNA vaccine would cover a much broader spectrum of HIV variants. From a technical point of view, the preparation of the DNA mixture is easier. A prerequisite for the DNA vaccine is a gp120 expression vector which carries the two interfaces BstEII and BamHI. This enables the conversion of the V3-Loop and V2-Loop variables env gene fragments into such a vector.

With regard to the GP120 proteins, it should be noted that in Area of the V3 loop 5 potential sites for N-glycosylation are located. The sugar residues protect the V3 loop from the Detection by neutralizing antibodies. Virus variants which have a glycosylated V3 loop are worse neutralizable as virus variants in which the glycosylation is incomplete. Since virus variants with a complete V3 loop glycosylation of the neutralizing immune response can escape, they are for transmission and for establishing the infection is important. In the course of the disease, if part of the neutralizing antibody response has been lost, virus variants prevail whose V3 loop is not fully glycosylated. Because the V3 loop is no longer covered by sugar residues, they replicate Virus variants faster than the fully glycosylated V3 loop Mutants. According to the invention it is therefore advantageous to the construction of the GP120 vaccine Consider glycosylation of the GP120 V3 loop.

In the context of the present invention, that shown in HIV Principle on other viruses that are in the course of viral Disease also develop variants, are transmitted. In particular, vaccines can be used against a variety of viruses be provided that have a broad immune response against many Induce virus variants in humans, which ideally against protects all variants. In therapy with such vaccines can the loss of neutralizing antibodies and the Effective loss of HIV-specific cellular immune response be fought. In this context it is also possible or even advisable for pathogen types (viruses) that develop in the course form sequence variants of several proteins of a disease, to provide several gene cassettes according to the invention, the each for variants of each of these proteins or fragments contain the same coding nucleic acid sequence to the Loss of neutralizing antibodies against all possible Counteract virus variants.

In the context of the present invention, the advantages of a protein vaccine and a DNA vaccine can advantageously be combined in order to increase the success of a preventive and / or therapeutic treatment of a viral disease. A pharmaceutical composition for the prevention and / or therapy of a virus infection is thus also provided, which comprises a protein mixture and a nucleic acid mixture, the protein mixture comprising ≥ 10 ² sequence variants of a viral protein or a part thereof and by Expression of a plasmid-DNA mixture is available, which has randomly distributed sequence combinations due to the variation of nucleotide positions, and wherein the nucleic acid mixture comprises ≥ 10 ² DNA molecules which code for sequence variants of a viral protein or a part thereof, the nucleic acid mixture due to the variation Sequence combinations randomly distributed from nucleotide positions. In particular, the pharmaceutical composition is a combination preparation which comprises both a protein mixture comprising one of the above-mentioned sequence variants of the GP120 protein and also one of the above-mentioned ones from the env sequence in SEQ ID NO: 1 or SEQ ID NO: 11 or a fragment of the same derived nucleic acid mixture.

The present invention is described below using examples, Figures and a sequence listing explained.

EXAMPLES

1.1 Klonierung von V3-loop kodierenden Oligonukleotiden

1.2 Klonierung der V3-env-Varianten

1.3 Analyse der Variabilität

1. General description of the manufacturing process

1.1 Cloning of V3-loop encoding oligonucleotides

1.2 Cloning of the V3 env variants

1.3 Analysis of variability

2.1 Abkürzungen

2.1.1 Allgemeine Abkürzungen

2.1.2 Nukleinsäuren

2.2 Bakterienstämme

2.3 Plasmide

2.4 Enzyme

2.5 Chemikalien

2.6 Oligonukleotide

2.7 Molekulargewichtsstandards

2.8 Verwendete Reagenzien-Kits

2.9 Medien

2.10 Sterilisieren von Lösungen

2.11 Anzucht und Lagerung von Bakterien

2.12 Herstellung kompetenter Zellen

2.13 Transformation in E.coli

2.14 Plasmid-DNA Präparation

2.15 Auftrennung von DNA in Agarosegelen

2.16 Reinigung von DNA aus Agarosegelen

2.17 Auftrennung von DNA in Polyacrylamidgelen

2.18 Schneiden von DNA

2.19 Ligation von DNA

2.20 DNA-Sequenzierung

2.21 Herstellung doppelsträngiger DNA mit Hilfe von Oligonukleotiden

2.22 Transfektion von COS-Zellen und CHO-Zellen

2.23 Chromatographische Reinigung der GP120-Mischung

2.24 Herstellung der DNA-Vakzine

2. Material and methods

2.1 Abbreviations

2.1.1 General abbreviations

2.1.2 nucleic acids

2.2 bacterial strains

2.3 Plasmids

2.4 enzymes

2.5 chemicals

2.6 oligonucleotides

2.7 Molecular Weight Standards

2.8 Reagent kits used

2.9 Media

2.10 Sterilize solutions

2.11 Cultivation and storage of bacteria

2.12 Production of competent cells

2.13 Transformation in E.coli

2.14 Plasmid DNA preparation

2.15 Separation of DNA in agarose gels

2.16 Purification of DNA from Agarose Gels

2.17 Separation of DNA in polyacrylamide gels

2.18 Cutting DNA

2.19 Ligation of DNA

2.20 DNA sequencing

2.21 Production of double-stranded DNA using oligonucleotides

2.22 Transfection of COS cells and CHO cells

2.23 Chromatographic purification of the GP120 mixture

2.24 Preparation of the DNA vaccine

1. General description of the manufacturing process 1.1 Cloning a mixture of V3-loop gp120 variants

The oligonucleotides are synthesized as described under 2.6. A mutated sequence is an example of a V3 loop sequence of the HIV-1 patient isolate F1-01 (M. Schreiber et al., J. Virol. 68 No. 6 (1994) 3908-3916). Any other sequence or Mixing sequences is also possible. To many different ones To clone variants of a V3 loop sequence at certain positions in the sequence instead of pure Nucleotide building block mixtures used. That way one a mixture of oligonucleotides, all in the Differentiate sequence and thus for different V3 loops encode. Such oligonucleotide mixtures are also known as oligonucleotides with degenerate sequences. Starting from chemically synthesized oligonucleotides with degenerate Sequences becomes the coding for the different V3 loops Area shown.

An oligonucleotide in reading frame orientation (forward) is used for the env sequence section from the BglII interface to synthesized first degenerate position. A second oligonucleotide, in a complementary orientation (reverse) is accordingly the sequence from a position 15 bases before The beginning of the variable range is synthesized (method 2.6). By overlapping the 3 'region over a total length of 15 Bases are the hybridization of both oligonucleotides. In a subsequent reaction with DNA polymerase (e.g. Taq DNA polymerase or Klenow fragment) arises from the two hybridized Oligonucleotides a completely double-stranded DNA molecule (Method 2.21).

The DNA mixture thus obtained is used with the restriction endonucleases BglII and XbaI digested. The cloning of the DNA mixture takes place in the expression vector cut with BglII and XbaI ΔV3-pBSCenvV3 (methods 2.15 and 2.16).

This vector contains the coding sequence of gp160 of HIV-1 strain NL4-3 (IIIB). The NL4-3 env gene was manipulated so that it has the restriction sites BglII and XbaI as well as ApaLI, PstI and BclI only once. The region coding for the V3 loop, which lies between the interfaces BglII and XbaI, was removed and replaced by a 15 base pair sequence, which introduces an analytical interface for the enzyme AscI. The BglII and XbaI cut V3-loop DNA mixture is cloned into this vector (method 2.19). The loss of the AscI interface in the finished V3-loop pBSCenvV3 vector can be used for the selection of the V3-loop coding clones (method 2.18).

The resulting plasmid mixture is transformed into DH5α bacteria (method 2.12), a transformation rate of> 10 ⁵ should be achieved. This results in a gene library of V3 loop fragments with a size of approximately 10 ⁵ clones. The mixture should be analyzed by DNA sequencing (see 1.3). With the help of this method, a mixture of clones is obtained, all of which code for different V3-loop variants of the GP120 protein. This mixture of vectors serves as a starting product for the preparation of the protein mixture for use as a vaccine and as a starting product for use as a DNA vaccine.

For the production of the GP120 protein mixture, the pBSCenvV3 expression vectors transfected into COS cells and CHO cells (Method 2.22). The purification of the various GP120 proteins, the mixture of active ingredients, as in the literature described, carried out according to method 2.23.

1.2 Cloning a mixture of V2-loop gp120 variants

Variants of the V1-loop and V2-loop are made in the same way manufactured. The expression vector ΔV3-pBSCenvV3 has for this three additional restriction interfaces. The variation of the V1 loop is done by cloning into the ApaLI and PstI Interfaces. The V2-loop is varied by cloning into the PstI and BclI interfaces.

1.3 Analysis of variability

The V3 loop mixtures are analyzed by DNA sequencing (Method 2.20). The statistical selection of approx. 100-200 clones, their V1, V2 and V3 loop sequences determined should be. If all of these clones are different, use With the help of a statistical calculation the heterogeneity of the Gene library and thus also the heterogeneity of the protein mixture be determined.

2. Material and methods 2.1 Abbreviations 2.1.1 General abbreviations

ug micrograms ul microliter .mu.mol micromoles APS ammonium persulfate bp base pairs dNTP deoxynucleoside triphosphate DNA deoxyribonucleic acid DTT dithiothreitol EDTA ethylenediaminetetraacetic G gram GP glycoprotein H hour HIV Human immunodeficiency virus IPTG Isopropyl-β-D-thiogalactopyranoside PUG 3-morpholino propane OD Optical density PAGE Poly-acrylamide gel electrophoresis PCR Polymerase chain reaction RT room temperature TEMED N, N, N ', N'-tetramethylethylenediamine

2.1.2 nucleic acids

A adenine C cytosine G guanine T thymidine

2.2 bacterial strains

Escherichia coli DH5α: F-, endA1, hsdr17, (rk-mk +), supE44, recA1, λ-, gyrA96, relA1, Φ80d lac z ΔM15

2.3 Plasmids

pBSCenvATG Own construction, the sequence is given in SEQ ID NO: 10.

2.4 enzymes

restriction enzymes MBI-Fermentas, Gibco-BRL, Biolabs DNA polymerases MBI Fermentas, Gibco BRL T4 DNA ligase MBI Fermentas

2.5 chemicals

[Α-35S] dATP Amersham Life Science Agarose, ultra pure GIBCO BRL ammonium persulfate Merck ampicillin U.S. Biochemical Bacto agar Becton Dickinson Bactotryptone Becton Dickinson boric acid Merck bromophenol Merck calcium chloride Merck deoxyribonucleotides MBI Fermentas dithiothreitol Biotechnology, St. Leon-Rot glacial acetic acid Merck ethidium bromide Sigma ethylenediaminetetraacetic Merck glycerin Merck urea ICN Biomedicals yeast extract Becton Dickinson potassium chloride Merck potassium Merck magnesium chloride Merck Acrylamide mix Roth sodium Merck sodium chloride Merck disodium hydrogen phosphate Merck sodium Merck 2-propanol Merck Sigmacote (chlorinated polysiloxane) Sigma N, N, N ', N'-tetramethylethylenediamine Merck Tris (hydroxymethyl) aminomethane GIBCO BRL

2.6 oligonucleotides

All oligonucleotides listed were with the Expedite ™ Nucleic Acid Synthesis System from PE Biosystems (Weiterstadt) manufactured. For the cloning of env genes that are only differ in the sequence for the V3 loop, oligonucleotides used. Using the example of cloning the V3 loop Region for the HIV-1 patient isolate F1-01 are the sequences of Oligonucleotides listed.

V3 loop: for cloning in pNL4-3 / BgIII-NheI, F1-01, forward:

V3 loop: for cloning in pNL4-3 / BglII-NheI, F1-01:

The oligonucleotides 7010, 7011 and the M13 standard primers (M13, M13r) were used for the sequencing of V3 loop clones.

2.7 Molecular Weight Standards

1 kb ladder MBI Fermentas 100 bp ladder MBI Fermentas

2.8 Reagent kits used

T7 sequencing kit Pharmacia Qiaquick PCR Purification Kit Qiagen Qiagen plasmid kit Qiagen

2.9 Media

YT medium: 10 g tryptone 5 g yeast extract 5 g NaCl dYT Media: 16 g tryptone 10 g yeast extract 5 g NaCl dYT agar plates: 10 g tryptone 5 g yeast extract 5 g NaCl 15 g agar

The amounts given refer to 1000 ml of deionized water. The batches were autoclaved at 121 ° C. and 1.5 bar for 20 min. If the media contained antibiotics or other heat-sensitive reagents, the appropriate amounts were sterile filtered and added after the media had cooled. ampicillin 3.3 ml / l (60 mg / ml) IPTG 3 ml / l (100 mM) Xgal 3 ml / l (2% in DMF, do not filter)

2.10 Sterilize solutions and devices

Solutions and media as well as pipette tips, Eppendorf tubes and Glassware was autoclaved at 121 ° C and 1.5 bar for 20-40 min. Heat sensitive solutions such as Antibiotic and IPTG solutions were sterile filtered.

2.11 Cultivation and storage of bacteria

Bacterial cultures were each with a single colony inoculated. To isolate a single colony, cells from one Spread liquid culture on an agar plate. After incubation isolated colonies grew at 37 ° C overnight. to The agar plates were kept for short-term storage of the bacteria sealed with parafilm and stored at 4 ° C. For a permanent Storage of bacterial strains was 0.75 ml of an YT overnight culture mixed with 0.25 ml sterile glycerin, in liquid Nitrogen snap frozen and stored at -70 ° C.

2.12 Production of competent cells

100 ml of YT medium were inoculated with 100 μl of an overnight culture. The bacteria were incubated in an shaking incubator at 37 ° C. until an OD ₅₆₀ = 0.4 and then centrifuged at 4 ° C. and 1000 g for 8 min. All subsequent work was done on ice using pre-cooled vessels and 4 ° C cold solutions. The cells were resuspended in 50 ml of 50 mM CaCl ₂ and incubated on ice for 30 min. After centrifuging again, the bacteria were taken up in 2.5 ml sterile TFBII buffer and divided into portions of 100 μl each. The 100 µl portions of the competent cells, if not required for immediate transformation, could be stored in liquid nitrogen at -70 ° C after shock freezing. TFBII buffer: 10 mM MOPS pH 7.0 75 mM CaCl ₂ 10 mM KCl 15% glycerin

2.13 Transformation of E. coli

(Hanahan, J. Mol. Biol. 166 (1983) 557-580)
A 100 ul portion of competent cells was thawed in an ice bath and incubated with 1-100 ng plasmid DNA for 30 min at 0 ° C. The transformation mixture was then incubated at 42 ° C. for 1 min. Then 1 ml of YT medium was added and shaken at 37 ° C. for one hour. In order to enable selection of the transformed cells, 100-500 μl of the mixture were spread on YT agar plates containing antibiotics. After overnight incubation at 37 ° C, only colonies that carry the plasmid-encoded resistance gene have grown. When transforming cells that do not contain a functional β-galactosidase (lacZΔM15 mutation, e.g. DH5α), vectors such as the pUC used enable a direct selection ( blue white screening) of recombinant bacteria via a plasmid-encoded β-galactosidase. For the blue-white selection, the bacteria were spread on Amp / IPTG / Xgal-YT agar plates. The substrate Xgal is split into a blue dye by the β-galactosidase. Due to the destruction of the reading frame of the lacZ gene by the insertion of a foreign DNA fragment into the lacZ gene, white colonies are generated. In contrast, blue colonies mean that the lacZ gene remained functional during cloning and ligation and that no DNA was inserted.

2.14 Plasmid DNA preparation

(Quiagen, Hilden)
The QIAprep Spin Miniprep Kit from Qiagen was used to isolate plasmid DNA from E. coli . The DNA preparation was carried out according to the manufacturer's instructions (QIAprep Plasmid Handbook 03/95). Plasmid-containing E. coli bacteria were inoculated in dYT-amp medium and shaken at 37 ° C. overnight. Three ml of the E. coli overnight culture were used for plasmid isolation. The bacteria were harvested (1 min, 14,000 rpm, Heraeus centrifuge) and taken up in 250 μl P1 buffer. The bacteria were disrupted by alkaline lysis (250 μl, 0.2N NaOH / 1% SDS). The mixture was neutralized by adding 3M potassium acetate (350 μl, pH 5.5). The purification of the plasmid DNA is based on the selective binding of plasmid DNA to DEAE anion exchange columns. At the selected salt concentrations and pH conditions, the plasmid DNA binds to the DEAE matrix. The DEAE matrix was washed (750 ul, PE buffer, Qiagen). The plasmid DNA was then eluted with 50 ul H ₂ O and stored at -20 ° C.

In order to obtain plasmid DNA in large quantities (approx. 100 µg), 25 ml overnight culture used. For the purification of the DNA the QIAGEN Plasmid Midi Kit was used. The purification based on the "QIAprep Spin Miniprep Kit" principle described and was based on the manufacturer's instructions (QIAGEN Plasmid Purification Handbook 01/97).

2.15 Separation of DNA in agarose gels

DNA was separated by gel electrophoresis in agarose gels. Depending on the size of the fragments examined, 0.8-2% agarose was dissolved in TBE buffer (for analytical agarose gels) or TAE buffer (for preparative agarose gels) by boiling. After cooling to about 60 ° C., 1 μl of ethidium bromide (10 mg / ml) was added to the gel liquid and poured into a prepared gel bed with a comb inserted. The completely cooled gel was overlaid in an electrophoresis chamber with TBE or TAE buffer and the comb removed. The DNA samples were mixed with 1/5 vol. Application buffer and pipetted into the sample pockets. The fragments were separated at 5-10 volts / cm gel length for 0.5-2 h. For detection, the gel was photographed after UV electrophoresis. The molecular weights of the DNA bands were determined in comparison to running DNA molecular weight markers. TBE buffer: 100 mM Tris 100 mM boric acid 3 mM EDTA TAE buffer: 40 mM Tris 2 mM EDTA 0.114% glacial acetic acid Order buffer: 0.25% bromophenol 0.25% xylenecyanol 30% glycerin 50 mM EDTA

2.16 Purification of DNA from Agarose Gels

The "QIAquick Gel Extraction Kit" (Qiagen, Hilden) was used to extract the DNA from agarose gels. The buffer conditions were chosen so that the nucleic acids bind to the silica membrane of the columns, while low-molecular bacterial components pass through the membrane (method 2.15). The "QIAquick Gel Extraction Kit Protocol" from the QIAqick Spin Handbook 07/97 was followed. The purified DNA was eluted from the silica membrane with 50 μl H ₂ O in each case.

2.17 Separation of DNA in polyacrylamide gels

The separation of radioactively labeled DNA fragments for DNA sequencing was carried out using denaturing polyacrylamide gels. Two cleaned glass plates were cleaned of grease residues with ethanol and coated with 1 ml Sigmacote per plate in order to obtain a hydrophobic surface. The coating should prevent the gel from tearing when pulled off the glass plate. Between the two glass plates the spacer (spacers) were placed, which served at the same time for the lateral sealing. The whole apparatus was fixed with several clips. To prepare the sequence gel, 21 g of urea were dissolved in about 20 ml of water. After adding 7.5 ml of acrylamide mix and 5 ml of 10x TBE buffer (see 2.15), the mixture was made up to 50 ml with water. The polymerization of the gel was started by adding 300 µl APS and 100 µl TEMED. Immediately afterwards, the gel solution was poured into the prepared gel apparatus and the pocket bottom was formed by inserting the flat back of the sawtooth comb. The gel was stored horizontally at room temperature until the polymerization was complete. After the gel had been inserted into the gel chamber, it was filled with TBE buffer. The job pockets were formed by turning the sawtooth comb over. In order to heat the gel to the optimal operating temperature, a lead time of 20 minutes was carried out. The bags were rinsed thoroughly with TBE buffer and then filled with 4-5 µl of the samples. The electrophoresis was carried out at 2 kV (150 watts) for about 2-3 hours. The gel was removed from the gel chamber after electrophoresis and one of the glass plates was carefully lifted off. The gel was transferred to Schleicher & Schuell paper (Whatman, England) by application and gentle pressure. After drying at 80 ° C. under vacuum, the gel was exposed to RT on an X-ray film (Kodak BioMax MR-1, Sigma Deisenhofen) for 1-3 days. Acrylamide mix: 40% polyacrylamide 0.8% bisacrylamide

2.18 Cutting DNA

Restriction endonucleases recognize and hydrolyze enzyme-specific palindromic DNA sequences, mostly 4-8 nucleotides in length. Depending on the type of enzyme, hydrolysis results in blunt ends or overhanging ends of single-stranded DNA ( sticky ends ). For a restriction digest, 0.1-60 μg of DNA were incubated with 2-120 units ( units ) of a restriction enzyme in the buffer specified by the manufacturer for 2-5 h at 37 ° C. The total volume was between 10 µl for analytical and 300 µl for preparative approaches. For restrictions with two different enzymes, a buffer was chosen in which both enzymes have sufficient activity, for example the EcoRI buffer for an EcoRI / BamHI digestion, or the conditions for the second enzyme were set after incubation with the first enzyme.

2.19 Ligation of DNA

DNA ligases catalyze the linking of DNA molecules using NAD ⁺ or ATP by forming a phosphodiester bond between a free 5'-phosphate group and a 3'-hydroxyl group. A three- to five-fold excess of DNA fragment was incubated with 100-500 ng cut plasmids and 5 units of T4-DNA ligase and 10 nmol ATP overnight at 12 ° C. in ligation buffer. Only cohesive end ligations were performed. Ligation buffer: 40 mM Tris-HCl pH 7.8 10 mM MgCl2 10 mM DTT 0.5 mM ATP

2.20 DNA sequencing

(Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467)
Sequencing of DNA was carried out using the chain termination method. Double-stranded plasmid DNA served as the substrate. Double-stranded DNA can be synthesized from single-stranded DNA starting from an oligonucleotide primer in the presence of dNTPs by a DNA polymerase. If a small proportion of dideoxynucleotides (ddNTPs) is contained in the nucleotide mixture, this leads to a randomly distributed incorporation of the ddNTPs, since the DNA polymerase cannot differentiate between dNTPs and ddNTPs. Due to the missing 3'-hydroxyl group of the ddNTPs, the incorporation leads to a termination of the double-strand synthesis and, since it is statistically distributed, to DNA strands of different lengths. Since the synthesis approach is divided into four and only one of the four ddNTPs is present in each sample, base-specific chain terminations occur in the respective approaches. To label the synthesized single strand, α- ³⁵ S-dATP is added to the reaction. After denaturation and separation of the DNA via polyacrylamide gel electrophoresis, the bands can be detected autoradiographically and the sequence of the DNA strand can be read directly.

The sequencing was carried out using the T7 sequencing kit (Pharmacia) according to the manufacturer's instructions. Either the M13 universal primer (Pharmacia) or primer M13r was used as the oligonucleotide primer. 32 µl (approx. 2 µg) of purified double-stranded plasmid DNA were denatured with 8 µl 2 M NaOH for 10 min at RT. After adding 7 µl 3 M sodium acetate pH 4.8, 4 µl H20 and 120 µl -20 ° C ethanol, the DNA was precipitated at -70 ° C for 20 min. The DNA was isolated by centrifugation (15 min, 4 ° C.), washed twice with cold 70% ethanol at −20 ° C. and dried in a vacuum centrifuge. The DNA sediment was then resuspended in 10 ul H ₂ O and after adding 2 ul annealing buffer and 2 ul primer solution (10 pmol in water) at 65 ° C for 5 min, 37 ° C for 10 min and 5 min at RT hybridized with the oligonucleotide primer. Immediately afterwards, 3 µl labeling mix, 1 µl α- ³⁵ S-dATP (1000 Ci / mmol, 10 µCi / µl) and 2 µl T7 polymerase solution (diluted 1: 5 with enzyme dilution buffer) were added for primer elongation and radioactive labeling and incubated for 5 min at RT. In each case 4.5 µl of this batch were placed on a MicroSample Plate (Greiner) preheated to 37 ° C, in which 2.5 µl of the four different dNTP / ddNTP mixes were present. After five minutes of incubation at 37 ° C, which was used for further elongation and base-specific termination, the reactions were stopped by adding 5 µl stop solution. Before application to the polyacrylamide gel, the samples were denatured for 2 min at 80 ° C. The samples were stored at -20 ° C. Annealing buffer: 1 st Tris-HCl pH 7.6 100 mM MgCl ₂ 160 mM DTT Labeling mix: 1.375 µM dCTP 1.375 µM dGTP 1.375 µM dTTP 333.5 mM NaCl Enzyme Dilution Buffer: 20 mM Tris-HCl pH 7.5 5 mM DTT 100 µg / ml BSA 5% glycerin Stop Solution: 10 mM EDTA 97.5% formamide 0.3% bromophenol 0.3% xylenecyanol A mix 840 µM dCTP C-Mix 840 µM dATP 840 µM dGTP 840 µM dGTP 840 µM dTTP 840 µM dTTP 93.5 µM dATP 93.5 µM dCTP 14 µM ddATP 14 µM ddCTP 40 mM Tris-HCl pH 7.6 40 mM Tris-HCl pH 7.6 50 mM NaCl 50 mM NaCl G-Mix 840 µM dATP T-Mix 840 µM dATP 840 µM dCTP 840 µM dCTP 840 µM dTTP 840 µM dGTP 93.5 µM dGTP 93.5 µM dTTP 14 µM ddGTP 14 µM ddTTP 40 mM Tris-HCl pH 7.6 40 mM Tris-HCl pH 7.6 50 mM NaCl 50 mM NaCl

2.21 Production of double-stranded DNA using oligonucleotides

The oligonucleotides were used to prepare the DNA mixture hybridized at 60 ° C. 100 pmol were used per oligonucleotide. 1 pmol of the hybridized sample was used for the PCR or 100 pmol used for the Klenow reaction.

The following cycles were carried out for the PCR: At the beginning 10 min 94 ° C 30 cycles 1 min 94 ° C 1 min 45 ° C 1 min 72 ° C At the end 10 min 72 ° C

1 pmol of hybridized oligonucleotides were used as the DNA template for the PCR. PCR primers were used in all PCR reactions carried out in a final concentration of 0.1 pmol / µl. 1 unit of the Taq polymerase was used per 50 μl of the reaction mixture. The concentration of the dNTPs in the PCR mixture was set to 0.1 mM.

For the preparation of the DNA mixture using the Klenow reaction, 100 pmol of the hybridized oligonucleotides were placed in Klenow buffer (50 mM Tris-HCl, pH 8.0; 50 mM MgCl ₂ , 10 mM DTT, 0.05 mM dNTP) solved. The reaction was started by adding 5 units of DNA polymerase I (Klenow fragment). After 30 min at 37 ° C the reaction was stopped by heating to 75 ° C (10 min).

2.22 Transfection of COS cells and CHO cells

5-10 µg linearized plasmid DNA are dissolved in 150 µl cell culture medium (without FCS and antibiotics). 30 μl of the transfection agent are added to the solution (SuperFect, Qiagen, Hilden). The mixture is incubated at RT for 10 min and added to the cells after the addition of 1 ml of medium. The COS cells (grown 80% confluent) were grown in 60 mm plates and washed with PBS just before transfection. After an incubation of 3 h at 37 ° C and 5% CO ₂ , the transfection medium was removed. The cells were then washed 4x with PBS and taken up in selection medium (DMEM, 5% FCS, 600µg / ml Geneticin).

2.23 Chromatographic purification of the GP120 mixture

1. Gelfiltration (Sephadex G-20)

2. Immunoaffinitätschromatographie
(anti-GP120-Antikörper gebunden an CH-Sepharose 4B)

3. Anreicherung durch Proteinfällung

4. Anreicherung durch Dialyse

The chromatographic purification is carried out according to standard methods (Techniques in HIV Research, Aldovini & Walker, Stockton Press, 1990; SW Pyle et al. Purification of 120.00 dalton envelope glycoprotein from culture fluids of human immunodeficiency virus (HIV) -infected H9 cells. AIDS Res Hum Retroviruses 1987, 3: 387-400). Cell extracts and cell culture supernatants from GP120-expressing COS cells are used for the isolation of the GP120 mixture. After centrifugation (25,000 g), the lysate is purified as follows:

1. Gel filtration (Sephadex G-20)

2. Immunoaffinity chromatography
(anti-GP120 antibody bound to CH-Sepharose 4B)

3. Enrichment through protein precipitation

4. Enrichment through dialysis

2.24 Preparation of the DNA vaccine

The starting material for the production of the DNA vaccine is the mixture of the BstEII-BamHI DNA fragments of the env gene, which were used for the production of the GP120-protein mixture. A eukaryotic expression vector for the HIV-1 GP120 which is approved for DNA vaccinations (see, for example, JD Boyer et al., J Infect Dis 1997, 176: 1501-1509; JD Boyer et al., Nat Med 1997, 3: 526 -532; ML Bagarazzi et al., J Med Primatol 1997, 26: 27-33) is changed so that the interfaces for BstEII and BamHI are located at identical positions on the gp120 reading grid in the env gene. The mutated, variable env gene fragments (BstEII-BamHI) are cloned into such a vector. The change of the env genes in the V2 loop and V3 loop area and their cloning in the DNA vaccine vector is carried out analogously to the cloning steps which are carried out for the preparation of the gp120 mixture.

SEQUENCE LISTING

<110> Strathmann AG & Co.

<120> Virus vaccine

<130> P052348

<140>
<141>

<150> 199 07 485.2
<151> 1999-02-12

<160> 12

<170> PatentIn Ver. 2.1

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Claims (50)

1. Protein vaccine which comprises a mixture of viral protein molecules, characterized in that the molecules are sequence variants of a single viral protein or of part of same, the mixture containing ≥ 10² sequence variants and being obtainable by expression of a plasmid-DNA mixture which comprises randomly distributed sequence combinations owing to variation in nucleotide positions.
2. Protein vaccine according to Claim 1, characterized in that the mixture contains ≥ 10³ and preferably ≥ 10⁴ sequence variants.
3. Protein vaccine according to Claim 1 or 2, characterized in that it comprises a mixture of GP120 proteins of HIV, which in each case differ from each other in their amino acid sequence in the region of the V2 loop and/or of the V3 loop.
4. DNA vaccine which codes for a mixture of structurally different virus proteins, characterized in that the vaccine contains a mixture of sequence variants of a viral DNA molecule or of part of same, the mixture containing ≥ 10² DNA molecules which differ from each other in their nucleic acid sequence, the mixture comprising randomly distributed sequence combinations owing to variation in nucleotide positions.
5. DNA vaccine according to Claim 4, characterized in that it contains a mixture of DNA molecules which code for sequence variants of a viral protein or of part.
6. DNA vaccine according to Claim 4 or 5, characterized in that the mixture contains ≥ 10³ and preferably ≥ 10⁴ DNA molecules which differ from each other in their nucleic acid sequence.
7. DNA vaccine according to one of Claims 4 to 6, characterized in that it codes for a mixture of structurally different GP120 proteins of HIV, in which the vaccine contains a mixture of DNA molecules, the nucleic acid sequences of which differ from each other in the region coding for the V2 loop and/or in the region coding for the V3 loop.
8. DNA vaccine according to Claim 7, characterized in that it contains a mixture of DNA molecules which differ from each other in their nucleic acid sequence such that they code for a mixture of GP120 proteins which contain amino acid sequences which differ from each other in the V2 loop and/or in the V3 loop.
9. Nucleic acid sequence which is derived from the env sequence represented in SEQ ID NO: 1 or from a fragment of same, characterized in that it is modified such that it contains ten monovalent restriction sites at intervals of about 150 base pairs.
10. Nucleic acid sequence according to Claim 9 which is derived from the env sequence represented in SEQ ID NO: 1 or from a fragment thereof, characterized in that it is modified such that it contains monovalent restriction sites which enable the specific exchange of the V2 and B3 regions.
11. Nucleic acid sequence according to Claim 9 or 10, characterized in that the sequence is modified by the introduction of silent mutations.
12. Nucleic acid sequence according to Claims 9 to 11, characterized in that it contains the sequence given in SEQ ID NO: 9.
13. Nucleic acid sequence, characterized in that it contains the sequence given in SEQ ID NO: 11.
14. Nucleic acid sequence, characterized in that it contains the sequence given in SEQ ID NO: 12.
15. Single-stranded nucleic acid sequence, which contains the region coding for the V3 loop and/or for the V2 loop of GP120, characterized in that

    a) in the V3 loop a 247 bp-long BglII-XbaI fragment or a 283 bp-long BglII-NheI fragment is exchanged for a modified fragment which, compared with the original fragment, comprises in either case inosine, a nucleic acid exchange or a mutation at 6 or more positions, and/or

    b) in the V2 loop a 139 bp-long PstI-BclI fragment or a 339 bp-long PstI-EcoRI fragment is exchanged for a modified fragment which, compared with the original fragment, comprises in either case inosine, a nucleic acid exchange or a mutation at 6 or more positions.
16. Nucleic acid sequence, characterized in that it is complementary to the sequence given in SEQ ID NO: 12.
17. Nucleic acid sequence according to Claim 15 or 16, characterized in that each or every fragment comprises inosine, a nucleic acid exchange or a mutation at 9 to 20 positions.
18. Double-stranded DNA which comprises hybrids of the single-stranded nucleic acid sequence according to Claim 15 or 17 with the single-stranded nucleic acid sequence according to Claim 16 or 17.
19. Nucleic acid mixture which comprises double-stranded DNAs, the nucleic acid sequences of which are derived from the env sequence in SEQ ID NO: 1 or SEQ ID NO: 11 or a fragment of same, characterized in that the nucleic acid sequences in each case differ from each other in the region coding for the V2 loop and/or in the region coding for the V3 loop, the mixture comprising randomly-distributed sequence combinations owing to variation in nucleotide positions and the nucleic acid sequences coding for a protein mixture which contains ≥ 10² sequence variants.
20. Nucleic acid mixture according to Claim 19, characterized in that the mixture contains ≥ 10³ and preferably ≥ 10⁴ sequence variants.
21. Protein mixture which comprises sequence variants of the GP120 protein, characterized in that it is a mixture of proteins which contain amino acid sequences which in each case differ from each other in the V2 loop and/or in the V3 loop, the mixture containing ≥ 10² sequence variants and being obtainable by expression of a plasmid-DNA mixture which comprises randomly distributed sequence combinations owing to variation in nucleotide positions.
22. Protein mixture according to Claim 21, characterized in that the mixture contains ≥ 10³ and preferably ≥ 10⁴ sequence variants.
23. Plasmid which contains an inserted double-stranded DNA according to Claim 18.
24. Expression vector, characterized in that it contains an inserted nucleic acid sequence according to Claims 9 to 14.
25. Expression vector according to Claim 24, characterized in that it contains the sequence given in SEQ ID NO: 10.
26. Expression vector, characterized in that it corresponds to DSM 12612.
27. Vector mixture which contains a mixture of plasmids according to Claim 23, characterized in that the nucleic acid sequences of the plasmids differ in each case from each other in the region coding for the V2 loop and/or in the region coding for the V3 loop, the plasmid mixture containing ≥ 10² plasmids and comprises randomly distributed sequence combinations owing to variation in nucleotide positions.
28. Vector mixture according to Claim 27, characterized in that the mixture contains ≥ 10³ and preferably ≥ 10⁴ plasmids which differ from each other in their nucleic acid sequence.
29. Vector mixture according to Claim 27 or 28, characterized in that the plasmids can be expressed in E. coli as host cell.
30. Vector mixture according to Claim 27 or 28, characterized in that the plasmids can be expressed in eukaryotic cells, preferably in Cos, CHO or BHK cells, as host cells.
31. E. coli host cells which are transfected with a vector mixture according to Claim 29.
32. Eukaryotic host cells which are transfected with a vector mixture according to Claim 30.
33. Eukaryotic host cells according to Claim 32, characterized in that they are host cells from the group consisting of Cos, BHK or CHO cells.
34. Process for the preparation of the nucleic acid sequence according to Claim 10, characterized in that so many silent mutations are introduced into a nucleic acid sequence coding for a viral protein that the nucleic acid sequence obtained as a result contains monovalent restriction sites which enable exchange of the V2 and V3 regions.
35. Process for the preparation of the nucleic acid sequence according to Claim 10, characterized in that so many silent mutations are introduced into a nucleic acid sequence coding for a viral protein that the nucleic acid sequence obtained as a result contains ten monovalent restriction sites at intervals of about 150 base pairs.
36. Process according to Claim 34 or 35, characterized in that the nucleic acid sequence coding for a viral protein is the sequence as per SEQ ID NO: 1 or SEQ ID NO: 11 or a fragment thereof.
37. Process for the preparation of the vector mixture according to Claims 29 and 30, characterized in that plasmids, the nucleic acid sequences of which in each case differ from each other in the region coding for the V2 loop and/or in the region coding for the V3 loop through random distribution of the bases at the varied nucleotide positions, are ligated into a vector which can be expressed in host cells.
38. Process according to Claim 37, characterized in that the host cells are E. coli, Cos, CHO or BHK cells.
39. Process for the preparation of the host cells according to Claim 31 or 32, characterized in that the host cells are transformed with a vector mixture according to Claim 27 or 28.
40. Process for the preparation of a protein vaccine according to one of Claims 1 to 13, characterized in that the host cells are cultivated according to one of Claims 31 to 33 under conditions which allow the expression of the mixture of viral protein sequence variants.
41. Process for the preparation of a DNA vaccine according to one of Claims 4 to 8, characterized in that the process is carried out according to Claim 37 or 38, the plasmids according to the invention being ligated into a vector which can be expressed in host cells of the organism to be vaccinated.
42. Use of a mixture of structurally different viral proteins which are sequence variants of a viral protein or of part of same, the mixture containing ≥ 10² sequence variants and being obtainable by expression of a plasmid-DNA mixture which comprises randomly distributed sequence combinations owing to variation in nucleotide positions, for the preparation of a vaccine for the prevention and/or therapy of a virus infection in humans.
43. Use of a protein mixture according to Claim 21 or 22 for the preparation of a vaccine for the prevention and/or therapy of an HIV infection in humans.
44. Use of a mixture of DNA molecules which code for ≥ 10² sequence variants of a viral protein or of part of same, the mixture comprising randomly distributed sequence combinations owing to variation in nucleotide positions, for the preparation of a vaccine for the prevention and/or therapy of a virus infection in humans.
45. Use of a nucleic acid mixture according to Claim 19 or 20 for the preparation of a vaccine for the prevention and/or therapy of a virus infection in humans.
46. Use of the nucleic acid mixture according to Claim 19 or 20 for the preparation of a vector mixture according to Claim 27 or 28 which can be expressed in host cells, the host cells being selected from the group consisting of E. coli, Cos, CHO and BHK cells.
47. Use of the vector mixture according to Claim 27 or 28 for the expression of a protein mixture according to Claim 21 or 22.
48. Use of the host cell according to one of Claims 31 to 33 for the preparation of a protein mixture according to Claim 21 or 22.
49. Pharmaceutical composition for the prevention and/or therapy of a virus infection, characterized in that it comprises a protein mixture and a nucleic acid mixture, the protein mixture comprising ≥ 10² sequence variants of a viral protein or of part of same and being obtainable by expression of a plasmid-DNA mixture which comprises randomly distributed sequence combinations owing to variation in nucleotide positions, and the nucleic acid mixture comprising ≥ 10² DNA molecules which code for sequence variants of a viral protein or of part of same, the nucleic acid mixture comprising randomly distributed sequence combinations owing to variation in nucleotide positions.
50. Pharmaceutical composition according to Claim 49, characterized in that it comprises a protein mixture according to Claim 21 or 22 and a nucleic acid mixture according to Claim 19 or 20.

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